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Edinburgh/11 August 2008
From 2008.igem.org
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Week 9
Monday 11 August 08
- PCR products of P55-P58 (cenA and cex) were run on Gel 41: cex with KOD polymerase (P57) generated a single band of the desired size. However, cex with Velocity polymerase and cenA with both KOD or Velocity polymerase failed. (Yan)
- PCR products P61-P63 (rrnB, cenA, cex) were run on Gel 42. Very clear band for rrnB, no bands for either cenA or cex. (CF)
- Made Plates 109-110 (Transformation of L33: pBS1A2+PcstA), 111-112 (transformation of L34: pBS1A2+dxs+crtE) and 113-114 (Transformation of L35: pBS1A2+dxs+lims1). (Yan)
- Made maxipreps X9 of pSB1A2+rbs+dxs (as for M72) and X10 of pSB1A2P+rbs+appY (as for M82). (AM)
- Redid PCR (KOD) of cex from C. fimi genomic DNA but with 10µl 50% glycerol, denaturing 1 min, annealing 60°C for 30s and extension 30s (P64). To be run on gel tomorrow. (AM)
- Digested P57 (cex from KOD PCR) with EcoRI/PstI to confirm identity of the gene. cex has a PstI restriction site near its midpoint, so this digestion should generate two segments of approximately 750bp. To be run on gel tomorrow. (AM)
- Digests performed using EcoRI/PstI to move crtE from BABEL2 (M79) to Edinbrick1. In each case, 3µl of vector was digested in 20µl total volume. The digests were run on a gel ready for excision, but gel showed no band corresponding to crtE (see gel 43 showing a single band at ~3kb for BABEL2+crtE and bands at ~2.5kb and 800bp for Edinbrick1). (AH)
- Minipreps M109-M112 (putative pSB1A2+crtB+crtI, plate 102), M113-M116 (pSB1A2+glgC-mut1,2, plate 103) and M117-M120 (pSB1A2+glgC-mut1,2,3, plate 94) were made. (OG)
- Double digests of M109-M120 with EcoRI/PstI were run on gel 44. M110-M112 (pSB1A2+crtB+crtI) have a band at 2.5kb as expected. M109 (pSB1A2+crtB+crtI) gave a band at 3kb instead, so should be ignored from now on. (Yan)
- Subbed white colonies from plates 105 (pSB1A2+crtY) and 106 (pSB1A2+rbs+crtY) to Plate 108. (CF)
